An optimized method for the chiral resolution of enantiomers of amino acids in bacterial supernatants is reported. This LC-MS/MS method is performed using a chiral Teichoplanin LC column and does not require sample clean up or chemical derivitization. This method allows for the determination of the relative amounts of the D and L enantiomers of 20 proteinogenic amino acids. The detection limits and response factors for the 20 amino acids were determined. Calibrations over three orders of magnitude showed least squares coefficient values (R^2) greater than 0.996 for eighty percent of the amino acids and greater than 0.992 for the remainder. The amino acids and their enantiomers were identified based on their retention times and their unique Multiple Reaction Monitoring (MRM) transitions for each amino acid. L-Aspartic acid-2,3,3-d3 was used as the internal standard. Cultures of Sinorhizobium meliloti (a nitrogen-fixing soil bacterium) were grown on minimal media; thus, all amino acids were biosynthesized by the bacterium. After centrifugation, supernatants were freeze dried, reconstituted in a small volume of methanol/water with internal standard and injected onto the LC column. The amino acids detected in the bacterial supernatant and the concentrations of the enantiomers were reported as the L and D isomers respectively: arginine [L, 12.6 ± 3.1 μg/L; D, 10.1 ± 3.2 μg/L], serine [L, 7.2 ± 1.16 μg/L; D, n.d.], threonine [L, n.d.; D, 11.2 ± 2.7 μg/L] and valine [L, 15.5 ± 4.3 μg/L; D, 11.3 ± 3.7 μg/L], where the term n.d. means below detection limit. The limits for detection for all amino acids ranged from 1.3 μg/L - 5.1 μg/L. In media with no added phosphate, the amino acid profiles changed somewhat under these stress conditions. Arginine was no longer detected while alanine and proline were now observed; the concentrations of the amino acids were: alanine [L, 7.7 ± 1.2 μg/L; D, 13.4 ± 2.5 μg/L], proline [L, n.d.; D, 8.63 ± 1.3 μg/L], serine [L, 7.6 ± 1.2 μg/L; D, n.d.], threonine [L, n.d.; D, 10.2 ± 3.2 μg/L] and valine [L, 11.6 ± 2.3 μg/L; D, 10.1 ± 3.1 μg/L]. These data represent the mean values of three independent bacterial growth experiments conducted over a 3 month period; the data came from the analysis of five separate aliquots from each growth experiment. The percent standard deviation for these data ranged from 15% to 33% and averaged 24%. Under both the normal and stressed growth conditions of S. meliloti produced the L enantiomer of serine, the D enantiomer of threonine and racemic valine. While racemic arginine was observed under normal growth conditions, levels were below detection under stressed conditions; under stress conditions only the D enantiomer of proline was observed while alanine was found in 1:2, L:D ratio. / Thesis / Master of Science (MSc)

Interações medicamentosas envolvendo a glicoproteína-P (P-gp) intestinal e placentária são determinantes na disposição cinética e transferência placentária de medicamentos durante a gestação. A fexofenadina, fármaco anti-histamínico, está disponível na clínica como racemato com indicação de uso durante a gravidez para o tratamento da rinite alérgica sazonal e urticária crônica. Considerando a fexofenadina como um substrato da P-gp e a fluoxetina, fármaco antidepressivo indicado durante a gravidez, um inibidor da P-gp, o presente estudo investiga a influência da fluoxetina na disposição cinética enantiosseletiva da fexofenadina em parturientes e suas relações com a transferência placentária in vivo e ex vivo. No estudo in vivo foram investigadas 16 parturientes, sendo 8 incluídas no grupo Controle e 8 incluídas no grupo Interação. Todas as parturientes investigadas receberam dose única oral de 60mg de fexofenadina racêmica, enquanto as parturientes do grupo Interação receberam também dose única oral de 40mg de fluoxetina racêmica 3 h antes da administração da fexofenadina. As amostras seriadas de sangue e urina foram colhidas até 48 h após a administração da fexofenadina. Na resolução do parto (2-3 h após a administração da fexofenadina) foram coletadas simultaneamente amostras de sangue materno, venoso e arterial do cordão umbilical e do espaço interviloso placentário. No modelo ex vivo, a farmacocinética transplacentária dos enantiômeros da fexofenadina foi avaliada em 4 lóbulos de placenta humana. Os enantiômeros da fexofenadina foram determinados nas amostras de plasma, urina e solução de perfusão placentária por LC-MS/MS acoplado a coluna de fase estacionária quiral Chirobiotic® V. A análise farmacocinética foi realizada empregando o programa WinNonlin e os testes estatísticos foram realizados com o auxílio do programa R. A disposição cinética da fexofenadina é enantiosseletiva no plasma materno com maiores valores de AUC0-? (423,20 vs 267,67 ng×h/mL) e menores valores de volume de distribuição aparente (621,37 vs 889,83 L), clearance total aparente (66,20 vs 105,05 L/h) e clearance renal aparente (5,25 vs 8,78 L/h) para o distômero (R)-(+)-fexofenadina. A transferência placentária da fexofenadina é limitada com razões de concentrações plasmáticas veia umbilical/veia materna de aproximadamente 0,16 para ambos os enantiômeros. As razões enantioméricas R-(+)/S-(-) de aproximadamente 1,7 nos compartimentos materno e fetal sugerem que a P-gp placentária não discrimina entre os enantiômeros da fexofenadina. A administração de dose única oral de 40 mg de fluoxetina racêmica 3 h antes da administração da fexofenadina aumentou os valores de AUC0-? (376,09 vs 267,67 ng×h/mL) e reduziu os valores de clearance total aparente (74,37 vs 105,05 L/h) e clearance renal aparente (3,50 vs 8,78 L/h) somente para o eutômero (S)-(-)-fexofenadina, inferindo inibição enantiosseletiva da P-gp intestinal. A administração de dose única oral de 40 mg de fluoxetina racêmica 3 h antes da administração da fexofenadina [concentrações plasmáticas materna no momento da extração fetal de 11, 9, 7 e 3 ng/mL, respectivamente para os enantiômeros (S)-(+)-fluoxetina, (R)-(-)-fluoxetina, (S)-(+)-norfluoxetina e (R)-(-)-norfluoxetina] não altera as razões de concentrações plasmáticas veia umbilical/veia materna e as razões enantioméricas R-(+)/S-(-) nos compartimentos materno e fetal. No modelo ex vivo, a transferência placentária da fexofenadina é lenta e limitada com razões de concentrações reservatório ii fetal/reservatório materno de aproximadamente 0,18 para ambos os enantiômeros. As razões enantioméricas R-(+)/S-(-) de aproximadamente 1,0 nos compartimentos materno e fetal confirmam que a P-gp placentária não discrimina entre os enantiômeros da fexofenadina. As concentrações clinicamente relevantes de 50 ng de cada enantiômero da fluoxetina/mL não alteram as razões de concentrações reservatório fetal/reservatório materno, a velocidade de transferência placentária e as razões enantioméricas R-(+)/S-(-) nos compartimentos materno e fetal. As razões de concentrações dos enantiômeros da fexofenadina reservatório fetal/reservatório materno obtidas no modelo ex vivo são similares às razões obtidas no estudo clínico de concentrações plasmáticas veia umbilical/veia materna, inferindo a validade do modelo ex vivo de predição da transferência placentária in vivo dos enantiômeros da fexofenadina. Concluindo, os estudos in vivo e ex vivo permitem inferir que a fluoxetina inibe de maneira enantiosseletiva a P-gp intestinal e não inibe a P-gp placentária em concentrações clinicamente relevantes / Drug-drug interaction on the intestinal and placental P-glycoprotein (P-gp) plays an important role in the kinetics disposition and placental transfer of drugs during pregnancy. Fexofenadine is an antihistamine drug for seasonal allergic rhinitis and chronic urticaria treatment during pregnancy and it is available as a racemic mixture. Taken together fexofenadine as a P-gp substrate and fluoxetine, antidepressant drug used in pregnancy, as a P-gp inhibitor, this study asses the effect of fluoxetine on the enantioselective kinetic disposition of fexofenadine in pregnant women at term and their relationships with in vivo and ex vivo transplacental transfer. The in vivo study investigated 16 parturients, 8 included in Control group and 8 included in Interaction group. All of parturients received 60 mg of racemic fexofenadine in a single oral dose, while Interaction group subjects were also given 40 mg of racemic fluoxetine in a single oral dose 3 h before fexofenadine administration. Serial blood and urine samples were collected for 48 h after fexofenadine administration. Maternal blood, venous and arterial umbilical cord blood, as well as placental intervillous space blood samples were simultaneously collected at delivery (2-3 h after fexofenadine administration). The transplacental pharmacokinetics of fexofenadine enantiomers was assayed in 4 placental lobule using ex vivo placental perfusion model. Fexofenadine enantiomers were determined in plasma, urine and placental perfusate samples by LC-MS/MS equipped with the chiral column Chirobiotic® V. Pharmacokinetic parameters were determined using WinNonlin and statistical analyses were performed using R statistical software. Fexofenadine kinetics disposition is enantioselective in maternal plasma with higher AUC0-? values (423.20 vs. 267.67 ng×h/mL) and lower apparent volume of distribution values (621.37 vs. 889.83 L), apparent total clearance (66.20 vs. 105.05 L/h) and apparent renal clearance (5.25 vs. 8.78 L/h) for (R)-(+)-fexofenadine distomer. Fexofenadine placental transfer is limited, with umbilical vein/maternal vein plasma concentration ratios of approximately 0.16 for both enantiomers. R-(+)/S-(-) enantiomeric ratios of approximately 1.7 in both maternal and fetal compartments indicate that placental P-gp might not have ability of fexofenadine\'s chiral discrimination. Single oral dose administration of 40 mg of racemic fluoxetine 3 h before fexofenadine administration increased AUC0-? values (376.09 vs. 267.67 ng×h/mL) and lowered both apparent total clearance values (74.37 vs. 105.05 L/h) and apparent renal clearance (3.50 vs. 8.78 L/h) only for (S)-(-)-fexofenadine eutomer, inferring intestinal P-gp enantioselective inhibition. Single oral dose administration of 40 mg of racemic fluoxetine 3 h before fexofenadine administration [maternal plasma concentrations at delivery of 11, 9, 7 and 3 ng/mL for (S)-(+)-fluoxetine, (R)-(-)-fluoxetine, (S)-(+)-norfluoxetine and (R)-(-)-norfluoxetine enantiomers, respectively] does not change either umbilical vein/maternal vein plasma concentration ratios or R-(+)/S-(-) enantiomeric ratios in maternal and fetal compartments. In the ex vivo model, placental transfer of fexofenadine is slow and limited, presenting fetal/maternal reservoirs concentration ratios of approximately 0.18 for both enantiomers. R-(+)/S-(-) enantiomeric ratios of approximately 1.0 on maternal and fetal compartments confirm placental P-gp does not have ability of fexofenadine\'s chiral discrimination. Clinically relevant concentrations of 50 ng of each fluoxetine enantiomer/mL neither alter fetal/maternal reservoirs concentration ratios, placental transfer rate nor R-(+)/S-(-) enantiomeric ratios in maternal and fetal iv compartments. Fetal/maternal reservoirs concentration ratios of fexofenadine enantiomers obtained in the ex vivo model are similar to those obtained in the clinical study of umbilical vein/maternal vein plasma concentrations, implying the validity of ex vivo model to predict placental transfer of fexofenadine enantiomers in vivo. In conclusion, both in vivo and ex vivo studies allow us to infer that fluoxetine enantioselectively inhibits intestinal P-gp and yet does not inhibit placental P-gp at clinically relevant concentrations.

O verapamil (VER) é um composto quiral comercializado como mistura racêmica dos enantiômeros (+)-(R)-VER e (-)-(S)-VER. O VER é biotransformado em norverapamil (NOR) e em outros metabólitos por vias dependentes do CYP. O tolueno e o n-hexano são solventes orgânicos que podem alterar o metabolismo de medicamentos dependente do CYP. Assim, o estudo investiga a estereosseletividade na farmacocinética do verapamil administrado a ratos na dose de 10 mg kg-1, sob forma racêmica, e do seu metabólito, norverapamil, bem como a influência do n-hexano e do tolueno na disposição cinética dos enantiômeros (+)-(R) e (-)-(S)-VER e (R)- e (S)-norverapamil em animais tratados com os solventes por inalação em câmara de exposição do tipo nose only nas concentrações de 88, 176 e 352 mg/m3 para o n-hexano e 94, 188 e 376 mg/m3 para o tolueno. Os enantiômeros do VER e do NOR foram separados na coluna de fase quiral Chiralpak® AD e analisados por LC-MS/MS (m/z = 441,3->165,5 para os enantiômeros do norverapamil e m/z 455,3->165,5 para os enantiômeros do verapamil). A análise farmacocinética foi realizada com base no modelo monocompartimental. A farmacocinética do verapamil é estereosseletiva em ratos do grupo controle não tratado com os solventes com acúmulo plasmático do eutômero (-)-(S)-VER (AUC0-? = 250,8 vs 120,4 ng mL-1 h; P<=0,05, teste de Wilcoxon). O metabólito (S)-NOR também foi acumulado no plasma dos animais do grupo controle com razão S/R relativa ao parâmetro AUC0-? de 1,5. Os parâmetros farmacocinéticos AUC0-?, Cl/F, Vd/F e t1/2 relativos aos enantiômeros (-)-S e (+)-(R)-VER e aos enantiômeros (S) e (R)-NOR não foram alterados pela exposição em câmara de exposição do tipo nose only ao n-hexano nas concentrações de 88, 176 e 352 mg/m3 e ao tolueno nas concentrações de 94, 188 e 376 mg/m3; teste de Kruskall-Wallis; P<=0,05. No entanto, a exposição ao n-hexano nas concentrações de 176 mg/m3 e 352 mg/m3 e ao tolueno nas concentrações de 94 mg/m3, 188 mg/m3 e 376 mg/m3 resultou em perda da enantiosseletividade observada para o grupo controle. / Verapamil (VER) is a chiral compound which is commercialized as a racemic mixture of the (+)-(R)-VER and (-)-(S)-VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP-dependent pathways. Toluene and n-hexane are organic solvents that can alter the metabolism of CYP-dependent drugs. The present study investigated the stereoselectivity in the pharmacokinetics of racemic VER administered to rats at a dose of 10 mg kg-1 and of its metabolite NOR. In addition, the influence of n-hexane and toluene on the kinetic disposition of the (+)-(R) and (-)-(S)-VER and (R)- and (S)-NOR enantiomers was analyzed in animals exposed by nose-only inhalation to n-hexane at concentrations of 88, 176 and 352 mg/m3 and to toluene at concentrations of 94, 188 and 376 mg/m3. The VER and NOR enantiomers were separated on a Chiralpak® AD chiral phase column and analyzed by LC-MS/MS (m/z = 441.3->165.5 for the NOR enantiomers and m/z 455.3->165.5 for the VER enantiomers). Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was stereoselective in control rats not treated with the solvents, with plasma accumulation of the (-)-(S)-VER eutomer (AUC0-? = 250.8 vs 120.4 ng mL-1 h; P<=0.05, Wilcoxon test). The (S)-NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC0-? ratio of 1.5. The pharmacokinetic parameters AUC0-?, Cl/F, Vd/F and t1/2 obtained for the (-)-S-VER, (+)-(R)-VER, (S)-NOR and (R)-NOR enantiomers were not altered by nose-only exposure to n-hexane at concentrations of 88, 176 and 352 mg/m3 or to toluene at concentrations of 94, 188 and 376 mg/m3 (P<=0.05), Kruskal-Wallis test). However, exposure to 176 and 352 mg/m3 n-hexane and to 94, 188 and 376 mg/m3 toluene resulted in the loss of enantioselectivity observed for the control group.

Orientador: Cesar Costapinto Santana / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-09-11T21:16:06Z (GMT). No. of bitstreams: 1 SilvaJunior_AbsolonCarvalhoda_M.pdf: 1195170 bytes, checksum: b6cb56c8b23987c514ae2eb904dc1b24 (MD5) Previous issue date: 2010 / Resumo: Alguns dos agentes terapêuticos são formulados e comercializados na forma de misturas racêmicas. O fármaco mitotano (o, p'- diclorodifenildicloroetano) é um exemplo dessa mistura. Utilizado apenas para o tratamento do carcinoma adrenocortical. A molécula do mitotano apresenta um carbono estereogênico, indicando a presença de dois enantiômeros. Estudos com o plasma de pacientes indicaram que os enantiômeros do mitotano apresentam perfis farmacocinéticos e farmacodinâmicos diferentes, motivando a separação dos enantiômeros para estudos pré-clínicos mais aprofundados. A separação cromatográfica dos enantiômeros do mitotano foi realizada, no presente estudo, em uma coluna empacotada com fase estacionária quiral tris 3-cloro-4-metil-dimetilfenilcarbamato de celulose e na tris-3,5 dimetilfenilcarbamato de amilose, comercialmente conhecidas como Lux Cellulose-2 e Chiralpak AD, respectivamente. Os experimentos foram realizados nas temperaturas de 15ºC, 25oC, 35ºC e 45ºC, com volume de injeção 20 µl numa concentração igual a 0,2 mg/ml na coluna Lux Cellulose-2 e 2 mg/mL na coluna Chiralpak AD, e vazões da fase móvel variando de 0,2 a 1,6 mL/min. Os parâmetros cromatográficos foram obtidos pelas seguintes fases móveis: (i) hexano puro, (ii) hexano/etanol 99,9:0,1% (v/v), e (iii) isopropanol/metanol 60:40% (v/v). Os fatores de retenção e a seletividade para a vazão de 1 mL/min com o uso de hexano puro como fase móvel e coluna Lux Cellulose-2 foram de 5,12; 5,81 e 1,14, respectivamente. Com a mesma vazão e com a adição de 0,1% em volume de etanol em hexano puro, os fatores de retenção foram de 1,30, 1,40, ao passo que o valor de seletividade obtida foi de 1,07. O tempo de retenção para a fase móvel contendo etanol foi de aproximadamente 8 minutos, que representa a metade dos valores obtidos com hexano puro, com um ganho substancial em tempo de processo de separação. Na coluna Chiralpak AD, utilizando-se a fase móvel isopropanol/metanol 60:40% (v/v), os fatores de retenção obtidos foram de 0,41, 1,35, enquanto que o valor de seletividade foi de 3,29. As curvas de Van Deemter foram obtidas nas duas fases estacionárias. As isotermas lineares e não-lineares foram determinadas. Também se realizaram as análises termodinâmicas da separação através das variações de entalpia, entropia e energias livre de Gibbs da adsorção, demonstrando estabilidade satisfatória das colunas / Abstract: Most therapeutic agents in the form of racemic mixtures are formulated and marketed. The drug mitotane (o, p'-dichlorodiphenyldichloroethane) is an example of this mixture, which is indicated as a single drug for treatment of adrenocortical carcinoma. Its structure has a carbon stereogenic, indicating the presence of two enantiomers. Studies with the plasma of patients indicate that the enantiomers of mitotane have different pharmacokinetic and pharmacodynamic profiles, motivating the separation of enantiomers for preclinical studies in greater depth. The chromatographic separation of these enantiomers in a packed column with chiral stationary phase cellulose tri-3-chloro-4-methyl-dimethylphenylcarbamate and amylose tris - 3, 5dimethylphenylcarbamate was performed. These species are commercially known as Lux Cellulose-2 and Chiralpak AD respectively. The experiments at temperatures of 15ºC, 25°C, °C and 45°C, with injection volume of 20µl at a concentration equal to 0.2mg/ml in Lux Cellulose-2 column and 2mg/mL in Chiralpak AD column, and a mobile phase flow rate ranging from 0.2 to 1.6mL/min were performed. The chromatographic parameters with mobile phases: (i) hexane, (ii) hexane/ethanol 99,9/0,1% (v/v) and (iii) isopropanol/methanol 60/40% (v/v) were obtained. The retention factors for the flow rate of 1mL/min using hexane as mobile phase and Lux Cellulose-2 column were 5.12, 5.81 respectively, showing a selectivity of 1.14. With addition of 0.1% of pure ethanol in hexane (v/v), for the same flow rate, the retention factors were 1.30 and 1.40 and selectivity of 1.07. The retention time for the mobile phase containing ethanol was approximately 8 minutes, which represents half of the value obtained with pure hexane, resulting a substantial gain in the time of the separation process. In Chiralpak AD column and a mobile phase of isopropanol/methanol 60/40% (v/v), the retention factors were 0.41, 1.35 with a selectivity of 3.29. The Van Deemter curve in the two stationary phases was obtained. The isotherms linear and nonlinear were determined. Thermodynamic analysis of the separation was performed, through variations of enthalpy, entropy and Gibbs free energy of adsorption, demonstrating through these analysis, one satisfactory stability of the columns / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

O nebivolol é um fármaco anti-hipertensivo, comercializado na forma de uma mistura equimolar dos enantiômeros RSSS e SRRR-nebivolol. Esses dois enantiômeros possuem propriedades farmacológicas distintas, o que sugere uma farmacocinética diferente entre ambos. Sendo assim, foram desenvolvidos dois métodos para a análise do nebivolol por eletroforese capilar (CE), um aquiral e outro quiral. O primeiro consistiu em um método para análise de forma farmacêutica comercial de comprimidos de nebivolol. Foram definidas as seguintes condições analíticas: capilar de sílica fundida 50 ?m de diâmetro interno (d.i) e 38 cm de comprimento efetivo (c.ef), eletrólito de corrida composto por tampão acetato de sódio 50 mM, pH 4,0, temperatura de 30 °C, tensão de 30 kV e detecção a 200 nm. O método desenvolvido permitiu a análise rápida e eficiente de comprimidos comerciais de nebivolol, sendo simples, seletivo, preciso e exato, está em conformidade com o guia de validação de métodos analíticos da ANVISA (2003), e foi aplicado para quantificação de comprimidos comerciais de nebivolol. O segundo método teve como objetivo realizar a análise enantiosseletiva da ligação do nebivolol à albumina humana do soro (HSA). Após a avaliação de diversos parâmetros, foram estabelecidas as seguintes condições analíticas: capilar de sílica fundida 50 ?m d.i e 38 cm c.ef, eletrólito de corrida composto por tampão acetato de sódio 50 mM, carboxi-metil-?-ciclodextrina 12,5 mM, 1% acetonitrila, pH 4,0, temperatura de 25 oC, tensão de 25 kV e detecção a 200 nm; e como técnica de stacking foi utilizada field amplified sample injection com plug de água (5 psi, 5 s) e injeção eletrocinética 5 kV por 30 s. Nesta condição foi obtida uma resolução de 1,58 e tempo de análise inferior a 25 minutos. No estudo de ligação à HSA foi observado que há enantiosseletividade na ligação, porém, este estudo ainda precisa ser melhor delineado em relação às concentrações de proteína e analito, bem como tempo de incubação e procedimento de filtração para separação das frações livre e ligada / Nebivolol is an anti-hypertensive drug, commercialized as a racemic mixture of RSSS and SRRR-nebivolol. Both enantiomers have distintict pharmacological properties, what suggests a different pharmacokinectis between them. Therefore, it was developed two methods for the nebivolol analysis by capillary electropforesis (CE), one of them is achiral and the other is chiral. The first method aimed the analysis of tablets. The analytical conditions were determined: silica fused capillary 50 ?m internal diameter (i.d.) and 38 cm effective length (ef. l.), running electrolyte composed by 50 mM sodium acetate buffer, pH 4.0, 30 °C temperature, 0 kV applied voltage and 200 nm UV detection. The developed method allowed a quickly and efficient tablets analysis, being simple, selective, accurate and precise, and it is also in accordance with ANVISA (2003) analytical methods validation guide, and it was applied to the quantification of nebivolol in tablets. The second method aimmed to analyze the enantiosselective nebivolol binding to HSA. After the evaluation of many parameters, it was stabilished the following analytical conditions: 50 ?m i.d. and 38 cm ef.l. fused silica capillary, running electrolyte composed by 50 mM sodium acetate buffer, 12.5 mM carboxymethyl- ?-cyclodextrin, acetonytrile 1%, pH 4.0, 25 oC, 25 kV applied voltage and 200 nm UV detection; and as stacking technique it was applied field amplified sample injection with water plug(5 psi, 5 s) and 5 kV por 30 s eletrokinect injection. At this condition, it was possible to achive 1.58 resolution and less than 25 minutes of analysis. At the HSA binding study, it was observed an enantiosselectivity on the binding; however this study still needs better desing in conserne to analyte and protein concentration, as well as, incubation time and filtration proceadure to the separion of binded and free fractions.

O metoprolol, um fármaco aceito no tratamento da hipertensão durante a gestação, está disponível na clínica como mistura racêmica dos enantiômeros S-(-) e R-(+), embora o S-(-)-metoprolol seja considerado o eutômero em termos do bloqueio do receptor 1 adrenérgico. O presente estudo avalia a influência do Diabetes mellitus gestacional na disposição cinética e no metabolismo enantiosseletivos do metoprolol em parturientes hipertensas. As parturientes hipertensas investigadas (n=35) com idade gestacional de 35-42 semanas e fenotipadas como metabolizadoras extensivas tipo metoprolol, foram distribuídas nos grupos controle (n=24) ou portadoras de Diabetes mellitus gestacional (n=11). As parturientes foram tratadas com dose única oral de 100 mg de tartarato de metoprolol racêmico 1-11 h antes do parto. Foram coletadas amostras seriadas de sangue materno (0-24h) e no momento do parto foram coletados simultaneamente sangue materno, sangue do cordão umbilical e líquido amniótico. Os enantiômeros do metoprolol e seus metabólitos foram quantificados por LC-MS/MS ou por detecção por fluorescência. A disposição cinética do metoprolol é enantiosseletiva em parturientes hipertensas com observação de maiores concentrações plasmáticas (AUC0- 113,42 vs 62,65 ng.h/mL) e menor clearance total aparente (344,21 vs 623,14 L/h) para o eutômero S-(-)-metoprolol. A formação do metabólito -hidroximetoprolol também é estereosseletiva com favorecimento do novo centro quiral 1R (AUC0- 1R/1S=2,84). O favorecimento da formação do R-(+)-ácido O-desmetilmetoprolóico (AUC0- 2,77 vs 2,66 g.h/mL) explica o acúmulo plasmático do S-(-)-metoprolol. O Diabetes mellitus gestacional compensado prolonga o tmax para ambos os enantiômeros do metoprolol (1,5 vs 2,5 h) e ácido O-desmetilmetoprolóico (2,0 vs 3,5 h) e para todos os isômeros do -hidroximetoprolol (2,0 vs 3,0 h). O Diabetes mellitus gestacional compensado não altera as razões isoméricas de concentrações plasmáticas do metoprolol, -hidroximetoprolol e ácido O-desmetilmetoprolóico. As razões de concentrações líquido amniótico/plasma materno obtidas para ambos os enantiômeros do metoprolol (3,0 para o R-(+)-metoprolol e 3,2 para o S-(-)-metoprolol) e para os isômeros do -hidroximetoprolol (5,1 para o 1\'S,2R; 4,0 para o 1\'S,2S; 1,6 para o 1\'R,2R e 2,3 para o 1\'R,2S) evidenciam maiores concentrações dos fármacos no líquido amniótico do que no plasma materno. No entanto, os enantiômeros do ácido O-desmetilmetoprolóico atingem menores concentrações no líquido amniótico do que no plasma materno das parturientes hipertensas (líquido amniótico/plasma materno = 0,29 e 0,37 respectivamente para os enantiômeros R-(+)- e S-(-)). A distribuição transplacentária é próxima a 1 para ambos os enantiômeros do metoprolol e para todos os isômeros do -hidroximetoprolol e próxima a 0,8 para ambos os enantiômeros do ácido O-desmetilmetoprolóico em parturientes hipertensas. O Diabetes mellitus gestacional compensado reduz em aproximadamente 20% a distribuição transplacentária dos isômeros 1S,2S; 1R,2R; e 1R,2S--hidroximetoprolol mas não altera a distribuição dos enantiômeros do metoprolol. / Metoprolol is a drug accepted in the treatment of hypertension during pregnancy and it is clinically available as a racemic mixture of its enantiomers S-(-) and R-(+) metoprolol, although S-(-)-metoprolol is considered the eutomer responsible for 1 adrenergic receptor blockade.This study evaluates the influence of gestational Diabetes mellitus on the kinetic disposition and metabolism of metoprolol enantiomers in hypertensive parturients. The investigated parturients (n=35) presented gestational age within 35 to 42 weeks, were phenotyped as extensive metabolizers of metoprolol and were distributed in the control group (n=24) or in the gestational Diabetes mellitus group (n =11). The parturients were treated with single oral dose of 100 mg racemic metoprolol tartrate 1-11 h before delivery. Maternal blood samples were collected until 24h after drug administration, whereas maternal blood, umbilical cord blood and amniotic fluid were simultaneously collected at delivery. Metoprolol enantiomers and its metabolites were quantified by LC-MS/MS or by fluorescence detection. Kinetic disposition of metoprolol is enantioselective in hypertensive parturients with observation of higher plasma concentrations (AUC0- 113.42 vs 62.65 ng.h/mL) and lower apparent total clearance (344.21 vs 623.14 L/h) for the S-(-)-metoprolol eutomer. The formation of -hydroxymetoprolol metabolite is also stereoselective in favor of the new chiral center 1\'R (AUC0- 1\'R/1\'S = 2.84). The formation in favor of R-(+)-metoprolol acid metabolite (AUC0- 2.77 vs 2.66 g.h/mL) explains the plasma accumulation of S-(-)-metoprolol. Gestational Diabetes mellitus prolongs tmax for both metoprolol enantiomers (1.5 vs 2.5 h), metoprolol acid metabolite (2.0 vs 3.5 h) and for all -hydroxymetoprolol isomers (2.0 vs 3.0 h). Gestational Diabetes mellitus does not alter the isomeric ratios of plasma concentrations of metoprolol, -hydroxymetoprolol and metoprolol acid metabolite. The concentrations of both metoprolol enantiomers (amniotic fluid/maternal plasma = 3.0 for R-(+)-metoprolol and 3.2 for the S-(-)-metoprolol) and -hydroxymetoprolol isomers (liquid amniotic fluid/maternal plasma = 5.1 for 1\'S,2R; 4.0 for 1\'S,2S; 1.6 for 1\'R,2R and 2.3 for 1\'R,2S) are higher in amniotic fluid than in maternal plasma. However, metoprolol acid metabolite enantiomers reach lower concentrations in amniotic fluid than in maternal plasma of hypertensive parturients (amniotic fluid/maternal plasma = 0.29 and 0.37 respectively for the R-(+)- and S-(-)- enantiomers). The transplacental distribution is approximately 1 for both enantiomers of metoprolol and all isomers of -hydroxymetoprolol and approximately 0.8 for both metoprolol acid metabolite enantiomers in hypertensive parturients. Gestational Diabetes mellitus reduces in approximately 20% the transplacental distribution of the isomers 1\'S,2S; 1\'R,2R and 1\'R,2S--hidroximetoprolol but does not alter the transplacental distribution of both metoprolol enantiomers.

<p>The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed.</p><p>The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. <i>N,N’</i>-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, <i>i.e.,</i> a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power.</p><p>A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.</p>

The thesis focuses on the development of new homogeneous gels for the separation of drug enantiomers, peptides, DNA and virus by electrophoresis and electrochromatography in capillaries and microchips. This type of separation media offers high resolution and small zone broadening. Compared to particulate beds the resolution in this type of separation media is high because the eddy diffusion is zero and the resistance to mass transfer is small, since the diffusional distance between two polymer chains in the gel is considerably shorter than that between two beads in a packed bed. The gels have been characterized in terms of plate heights, plate numbers, resolution, etc. Gels of agarose, polyvinyl alcohol, albumin and polyacrylamide have been employed for electrochromatography or electrophoresis. N,N’-methylene-bisacrylamide, the most widely used crosslinker in polyacrylamide gels, was exchanged for allyl-β-cyclodextrin to get a multi-purpose gel, i.e., a separation medium the separation properties of which is determined not only by the polyacrylamide chains, but also by β-cyclodextrin with its complexation power. A cost-effective, hybrid microdevice has been designed for fast electrophoretic and electrochromatographic analyses as well as for microchromatography. It consists of a fused silica capillary mounted on a supporting plate which integrates most of the compartments necessary for automation and sensitive detection at short UV wavelengths.

The research work reported in this thesis deals with the development of novel NMR experimental techniques for the spectroscopic discrimination of enantiomers dissolved in a chiral liquid crystalline medium. The information on the chemical shifts and coupling constants pertaining to each enantiomer has been derived on the investigated chiral molecules. The enantiomeric excess (ee), a parameter which is of profound importance in pharmaceutical industry and in asymmetric synthesis, has also been measured. A special attention is paid to the use of high sensitivity of H NMR for chiral discrimination. Typical analyses of H NMR spectra are severely hindered due to enormous spectral inhomogeneous broadening arising from too many unresolved transitions, in addition to superposition of spectra from both the enantiomers. Therefore, the major part of the work is focused on the design and application of pulse sequences to overcome many of these drawbacks. This helps to achieve very high resolution, discerning of overlapped transitions, identification of resonances pertaining to each enantiomer and simplification of the spectrum for easy extraction of spectral parameters, in addition to the accurate measurement of ee. Initially a brief discussion is provided on enantiomers, diastereomers, basic principles of NMR spectroscopy, the several interaction Hamiltonians responsible for yielding the NMR spectra, introduction to product and polarization operator formalisms that gives insight into the spin dynamics for designing appropriate two-dimensional (2D) NMR experiments. This sets the foundation to understand the complex multiplet structures of the diagonal peaks and cross peaks in the resulting 2D spectrum. Subsequently, a brief introduction is given for the available techniques for NMR spectroscopic discrimination of enantiomers in isotropic medium, where only chemical shifts are employed as a measurable parameter. The limitations of these techniques are circumvented by the introduction of other anisotropic NMR parameters, such as homo-and hetero-nuclear dipolar couplings, quadrupolar couplings and chemical shift anisotropies. To achieve this goal the enantiomers are dissolved in weakly aligning chiral liquid crystalline (CLC) medium. To understand this, a general introduction to liquid crystals and their utility as an alignment medium in NMR spectroscopy and the anisotropic interactions affecting the NMR spectrum has also been provided. The preparation of the CLC phase of Poly-γ-Benzyl-L-Glutamate (PBLG) employed in the present study and its orientational behaviour has been discussed. The detection of NMR spectra of various nuclei and the interaction parameters utilized for chiral discrimination will be enumerated. A brief summary of the experiments employed for the spectral analyses of the enantiomers dissolved in PBLG will also be presented.

The work described in this thesis involves the study of weak interactions by NMR spectroscopy and using them to develop novel applications. The two different applications chosen are i) using molecular interactions for chiral discrimination and ii) understanding the nature of the interaction between peptide and nanoparticles to develop potent antibacterial agents. The thesis, which is divided into five chapters starts with a general introduction of NMR spectroscopy for the study of molecular interactions in conjunction with other techniques. The remaining four chapters focus on four different areas/projects that I have worked on. Chapter 1: Introduction This chapter reviews different kinds of molecular interactions along with the introduction to NMR spectroscopy and other techniques used for all the studies. Starting with the application of chiral discrimination the chapter proceeds to the general introduction of antimicrobial peptides, silver nanoparticles and the strategy for peptide resonance assignment. Chapter 2: Chiral discrimination for versatile functionalities There are many chiral agents available for discriminating enantiomers which mainly target specific functional groups. In this study, we have explored a strategy involving ternary complexation to investigate chiral discrimination of different kind of functional groups by NMR spectroscopy. The proposed protocol was employed for the enantiodiscrimination of molecules containing functional groups, such as amino alcohols, secondary alcohols, cyanohydrins, oxazolidones, diols, thiones and epoxides, using a phosphorous based three component mixture. The simple mixing and shaking of enantiopure 1,1’-binaphthyl-2,2’-diyl hydrogenphosphate (BNPA), 4-(dimethylamino)pyridine (DMAP) and a chiral analyte in the solvent CDCl3 served as a chiral solvating agent and resulted in well-dispersed peaks for each enantiomer in the 1H NMR spectrum. Discrimination was achieved not only for the proton at the chiral center but also for multiple proton sites. The J-resolved technique was used for alleviating the spectral complexity pattern to accurately measure the chemical shift difference. The devised approach also permitted the precise measurement of the enantiomeric excess (ee). Chapter 3: Simultaneous discrimination of secondary alcohols and carboxylic acids In this chapter, I describe two novel ternary ion-pair complexes, which serve as chiral solvating agents (CSA), for enantio discrimination of secondary alcohols and carboxylic acids. The superiority of CSA over other auxiliaries arises due to the formation of diastereomeric complexes through non-covalent interactions with the analyte. By exploiting the acid-base interaction strategy and employing DMAP, which further enhanced the hydrogen bonding efficiency the discrimination for both carboxylic acids and secondary alcohols were achieved. The protocol for discrimination of secondary alcohols is designed by using one equivalent mixture each of enantiopure mandelic acid, 4-dimethylaminopyridine (DMAP) and a chiral alcohol. For discrimination of carboxylic acids, the ternary complex is obtained by one equivalent mixture each of enantiopure chiral alcohol, DMAP, and a carboxylic acid. Furthermore, the formation of the complex was supported by calculating the energy-minimized structure of the proposed complex by density functional theory (DFT). The designed protocols also permit accurate measurement of the enantiomeric composition. Chapter 4: Enhanced potency of nanoparticle-antimicrobial peptide conjugates Antibiotic resistance is emerging as the new global health problem. Due to the blatant misuse and overuse of these drugs has resulted in the bacteria becoming resistant to a wide range of antibiotics. Researchers have found an alternative of current antibiotics which are a group of peptides known as antimicrobial peptides (AMP). But using these molecules as drug is rather costly due to high synthesis cost. Further the antibacterial activity of silver nanoparticle is well established. However, due to its toxic nature after, it cannot be used in high concentration. The conjugation of nanoparticles with antimicrobial peptides is emerging as a promising route to achieve superior anti-microbial activity. However, the nature of peptide-nanoparticle interactions in these systems remains unclear. This study describes the interactions of antimicrobial peptide with silver nanoparticles by NMR spectroscopy in conjunction with other biophysical techniques to completely understand the underlying mechanism of interaction between nanoparticles and peptide. It reveals that the conjugation process involves dynamic interaction between the nanoparticle and the peptide. This study also confirms the enhanced antibacterial efficiency of the nano-conjugate towards bacterial killing compared to the nanoparticle or the peptide alone. Chapter 5: Mechanistic insights into the action of nano-conjugates It is well established that antimicrobial peptides act as pore-formers to rupture the bacterial cells. This chapter is focused on studying the mechanism of action of the nano-conjugate with bacterial membrane mimic models. This study for the first time reveals the details of nanoconjugate membrane interaction at an atomic level. The pore formation mechanism and the enhanced efficiency of the nanoconjugate were explored using fluorescence spectroscopy, CD spectroscopy, and NMR spectroscopy. Structural changes of the peptide and the nanoparticle bound peptide have been captured which infers the propensity of the peptide to form a helical structure upon interacting with the membrane. The calculated structure of the peptide and nanoparticle bound peptide remains almost identical in presence of the membrane mimic environment. In the case of the nanoconjugate, the increase in local positive charge concentration makes the system to penetrate the bacterial membrane faster which further allows the nanoparticle to access the intercellular organelles easily. This dual mode of mechanism thus makes this nano-conjugate a promising antibacterial agent towards multi drug resistant bacteria. In summary, the thesis has focused on the studies of weak intermolecular interactions in different chemical and biological systems using NMR spectroscopy. It is demonstrated that in certain chemical systems, such interactions can be exploited to discriminate enantiomers and determine the enantiopurity of compounds by NMR. In the case of biomolecules, such weak interactions exist when protein or peptides interact with nanoparticles. Using silver nanoparticles, it is shown that such interactions result in a stable conjugate system. NMR spectroscopy provides valuable insights into the structure and dynamics of the system. Further, by using anti-microbial peptides conjugated with silver nanoparticles, new superior antibacterial agents can be developed.